ABSTRACT Hematopoietic stem cells (HSCs) regenerate blood cells throughout life. Defects in HSC maintenance can lead to anemia, impaired immunity, bone marrow failure and cancer. A mechanistic understanding of HSCs is crucial for uncovering the factors that result in their dysfunction and harnessing their regenerative potential. We recently discovered that HSCs have lower rates of protein synthesis than other hematopoietic cells and that this is necessary for HSC maintenance, as genetic changes that increase protein synthesis impair HSC function. This raises a fundamental question of how cell-type specific differences in protein synthesis promote HSC function. In preliminary studies, we determined that HSCs exhibit superior protein quality as compared to restricted progenitors, and increasing protein synthesis reduces protein quality within HSCs. This raises the possibility that low protein synthesis promotes HSC function by enhancing protein homeostasis (proteostasis). In Aim 1, we will test if declines in protein quality impair HSC function. We will examine hematopoiesis, HSC function and aging in Aarssti/sti mice that have a tRNA editing defect that reduces translational fidelity leading to an accumulation of mistranslated proteins. A second question is how increased protein synthesis is sensed by HSCs and how it impairs their function. High rates of protein synthesis can increase translational errors that lead to protein misfolding. An accumulation of misfolded proteins can overwhelm the ubiquitin proteasome system (UPS). This raises the possibility that HSCs sense changes in protein synthesis via effects on the UPS. The UPS regulates HSC fate by modulating the turnover of several proteins, including c-Myc. We determined that increased protein synthesis promotes c-Myc accumulation in HSCs. In Aim 2 we will use transgenic UPS reporter mice to test if increased protein synthesis disrupts UPS activity within HSCs in vivo. We will breed Mx1-Cre+;Ptenfl/fl mice with Mycfl/+ mice and test if reducing c-Myc expression rescues Pten-deficient HSCs, which are normally depleted by increased protein synthesis. Finally, in Aim 3, we will examine the underlying molecular mechanisms that attenuate protein synthesis in HSCs. In preliminary studies we found that HSCs express low levels of Eif5 protein compared to progenitors. Eif5 promotes protein synthesis by stimulating 80S ribosome assembly. Interestingly, HSCs preferentially express a long isoform of Eif5 that contains a long 5?UTR predicted to form complex secondary structures that could impede its translation. We will test if differential splicing of Eif5 limits its translation and restricts protein synthesis in HSCs. We will overexpress Eif5 in HSCs and assess protein synthesis and HSC function. We will test the translational efficiency of Eif5 5?UTRs in luciferase reporter assays. Using a mouse that only expresses the short isoform of Eif5, we will test if differential splicing affects protein synthesis, proteostasis and HSC function in vivo. These studies could unravel a new mechanism whereby low protein synthesis enhances proteostasis to promote HSC function.